Recent advances in biotechnology and molecular biology offer tremendous opportunities to develop biotech organisms with commercially desirable characteristics or traits. In particular, modern genetic engineering techniques have greatly accelerated the introduction of new genes and hence new traits into recombinant cells and organisms, particularly microbial organisms. The proper expression of a desirable transgene in a transgenic organism is widely considered to be a requisite requirement to achieve this goal. For example, expression of a gene in a recombinant cell that does not normally express such a gene may confer a desirable phenotypic effect. In another example, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene. Moreover, for production of recombinant cells and organisms with various desired characteristics, it would be advantageous to have a variety of promoters to provide gene expression such that a gene sequence can be transcribed efficiently in the amount necessary to produce the desired effect.
Furthermore, as the field of microbial transgenesis rapidly develops and more genes become accessible, a greater need exists for microorganisms transformed with multiple genes. In fact, the commercial development of genetically improved organisms has advanced to the stage of introducing multiple heterologous genes and traits into a single recombinant cell. These multiple heterologous genes typically need to be transcriptionally controlled by diverse regulatory sequences. For example, some transgenes need to be expressed in a constitutive manner whereas other genes should be expressed at certain developmental stages or in specific compartments of the transgenic cell. In addition, multiple regulatory sequences may be needed in order to avoid undesirable molecular interactions which can result from using the same regulatory sequence to control more than one transgene. In light of these and other considerations, it is apparent that optimal control of gene expression and regulatory element diversity are important in modern recombinant biotechnology.
However, despite the availability of many molecular tools, the genetic modification of recombinant organisms is often constrained by an insufficient expression level or temporally nonspecific expression of the engineered transgenes. In addition, while previous technological advancements have provided a number of regulatory elements that can be used to affect gene expression in transgenic organisms, there is still a great need for novel regulatory elements with beneficial expression characteristics. One example of this is the need for regulatory elements capable of driving gene expression preferentially in different microbial growth phases. On the other hand, there also exists a continuing need for regulatory elements capable of driving gene expression constitutively throughout cell life cycle and/or unaffected by growth conditions, as well as at low, moderate, high, or very high transcription levels. Thus, the identification of novel molecular tools including genes, vectors, regulatory elements that function in various types of organisms and in distinct growth phases and growth conditions will be useful in developing genetically enhanced organisms.